|Fig. 1: Mobile pilot-plant fermentor used as a cellulosic ethanol bioreactor. (Source: Wikimedia Commons)|
Fossil fuels are a non-renewable energy resource whose combustion generates green house gases (GHG) like carbon dioxide. Despite recent strides to reduce GHG emissions, U.S. emissions have increased at an average rate of 0.3% per year since 1990.  While bioethanol has lower energy density than gasoline, a shift from fossil fuels towards bioethanol production can help mitigate greenhouse gas emissions.
The type of fermentation substrate differentiates the two primary bioethanol production methods: first generation utilizes grains and starches, while the second generation uses lignocellulose. Although first generation bioethanol production methods are currently the most popular in the U.S., it has some significant disadvantages. Corn-based bioethanol only reduces GHG emissions by 18% compared to gasoline, while cellulosic bioethanol reduces GHG emissions by 90%.  Another disadvantage of first generation biofuels is the food-versus-fuel debate: one of the reasons for growing food prices is the increased production of grain and starch-based bioethanol.  Lignocellulose consists of plant biomass from a variety of sources including forest and paper waste. The exact composition of lignocellulose varies by plant species due to genetic and environment influences, but it always contains three major components - cellulose, hemicellulose, and lignin.
Cellulose is the major component and comprises anywhere from 40-60% of the dry mass of lignocellulose.  It is a linear polymer of glucose molecules arranged in bundled fibrils, forming a sturdy crystalline matrix. Hemicellulose constitutes 20-40% of the dry mass of lignocellulose.  It is an extensively branched heteropolymer composed of a variety of pentoses and hexoses. Both cellulose and hemicellulose are polysaccharides that can be hydrolyzed and fermented into ethanol through biochemical pathways. Lignin accounts for the remaining 10-25% of the dry mass of lignocellulose and is a polymer of aromatic molecules organized into a tight matrix.  Unlike cellulose and hemicellulose, lignin is highly resistant to chemical and physical degradation. Its presence is one of the main drawbacks of using lignocellulose for bioethanol production. In order to effectively hydrolyze the cellulose and hemicellulose into fermentable sugars, they must be liberated from the dense lignin matrix encasing them in a process known as pretreatment. 
While a number of pretreatment methods have been developed, the most popular method utilizes acid- mediated hydrolysis. Dilute sulfuric acid hydrolyzes hemicellulose into glucose and releases cellulose from the lignin matrix. Despite its effectiveness, there are a number of disadvantages: acid corrosion of containers requires expensive repairs, residual acid must be neutralized before fermentation, and acid-mediated hydrolysis forms a number of degradation byproducts that need to be filtered out.  A subsequent dilute sulfuric acid treatment hydrolyzes the cellulose into glucose monomers. However, this two-step acid treatment forms degradation products such as tars, which cannot be fermented into ethanol. Overall, this two-step acid-mediated hydrolysis has a glucose recovery rate of only 50%.  The pretreatment phase is one of the main barriers to efficient second-generation bioethanol production from lignocellulose because it accounts for approximately 33% of the total cost of second generation bioethanol production. 
An alternative but more expensive method of glucose recovery from cellulose relies on enzymatic hydrolysis catalyzed by cellulase, which specifically converts cellulose to glucose with recovery yields as high as 85%.  However, this process is slower than acid-mediated hydrolysis because the lignin sheathe reduces cellulase accessibility to cellulose. Furthermore, cellulase cannot hydrolyze hemicellulose. Because a number of different sugar monomers comprise hemicellulose, a cocktail of enzymes is necessary for complete hydrolysis of hemicellulose as well. Overall, the slowness and cost of enzyme- mediated hydrolysis discourages its application at the industrial scale. 
Since the pretreatment phase is one of the main economic barriers to more cost-efficient second-generation bioethanol production, recent research has been focused on optimizing the process. One possible solution is changing the cellulose source. Certain species of bacteria can produce bacterial cellulose. Although plant and bacterial cellulose share the same molecular formula, bacterial cellulose lacks hemicellulose and lignin. Without these two components, the acid-catalyzed hydrolysis step can be avoided, reducing the cost of the pretreatment phase.  Furthermore, enzyme-mediated hydrolysis of cellulose will be accelerated due to the lack of lignin hindering accessibility to cellulose. The main barrier to implementation of industrial scale bacterial cellulose production is the cost of bacterial growth media.  The main advantage of using lignocellulose is the abundance of plant biomass in the form of byproducts and waste from crops. Use of bacterial cellulose increases the purity of the cellulose, but nullifies one of the main advantages of second generation bioethanol production - abundant starting material.
Growth media requires a carbon and nitrogen source. Currently, most growth media utilize sugars like glucose, fructose, and sucrose as the carbon source. Use of these sugars for growth media would defeat the purpose of bacterial cellulose production because fermentation of glucose is already the intended end-goal. Growth media consisting of sucrose yielded 4.9 g bacterial cellulose per L of media, for a total yield of 0.53 g of bacterial cellulose per g of sucrose.  This process is grossly inefficient because there is a return of only 53% on the carbon inputted.
In order to make bacterial cellulose production cost-effective, alternative growth media must be developed. One current avenue is using industrial waste as growth media. Tsouko et al. have found that the bacterial strain K. sucrofermentans DSM 15973 could produce relatively high concentrations of bacterial cellulose when cultivated in waste streams from oilseed-based biodiesel industries and confectionary industries.  Crude glycerol is a byproduct of biodiesel production. Using crude glycerol supplemented with waste from oilseed-based biodiesel industries or confectionary industries, yield of bacterial cellulose was increased to approximately 13 g of bacterial cellulose per L of media. The carbon return was 0.89 g of bacterial cellulose per g of crude glycerol. 
While less than 100% return on inputted carbon using crude glycerol and waste streams is not ideal, the process is still viable. Because the carbon source and supplement were both waste products from already existing industries, bioethanol produced from bacterial cellulose taps into carbon and cellulose reserves previously disregarded.
© Jeremy Uang. The author grants permission to copy, distribute and display this work in unaltered form, with attribution to the author, for noncommercial purposes only. All other rights, including commercial rights, are reserved to the author.
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